The Structure Optimization And Anti-inflammatory Mechanism Of Hydrostatin-SN1 From The Venom Of Sea Snake

Background The snake venom contain many complex peptides and protein.Most of them reveal comprehensive toxicological and pharmaceutical activity.The active ingredients and mechanisms of anti-inflammatory and immunosuppression are still unkown.The Chinese take the snake as traditional medicine to treat rheumatoid arthritis and other autoimmune diseases for hundrad years.The peptide isolated from venom can attenuateo xidative stress and decrease inflammatory chemokine.Our group take human recombinant tumor necrosis factor receptor?(hr TNFR1)as substrate to screen a T7 phage display library of Hydrophis cyanocinctus.We found a TNFR1 selective binding peptide,named as Hydrostatin-SN1.BIAcore assay showed SN1 can bind to two receptors.It inhibit TNF-?-mediated activation of NF-?B and MAPKs signaling in vitro.Hydrostatin-SN1 also can inhibit TNF-?-mediated inflammatory diseases in vivo animal models.Compare with the small molecule,the synthesis cost of SN1 is higher since it has more molecules and larger size.Compare with the traditional biologic drugs,SN1 has short half-life since it can be easily degradable and poor binding capacity.In order to elevate its anti-TNF activity and extend the half-life,we explore which amino acid binding to TNFR1 specificly and optimize its structure.Objective Clarify the amino acid of SN1 binding to TNFR1,reform the structure to elevate its activity and evaluate the anti-inflammatory effect of peptide in vitro and vivo.Methods 1.The bioinformatics of SN1 was analysed with online server.Using Builder program in Discovery Studio2.5 soft pakage to show peptide from scratch.ZDOCK is used to stimulate the structure of SN1 binding to TNFR1,the SN1-TNFR1 complex is optimsed by molecule dynamic.The core sequences of binding sites are determined.2.11 peptides were cutted from SN1.The inhibition of the peptides on TNF-?-induced cytotoxicity in L929 cells was assayed by MTT.The anti-inflammatory activity of short peptide is verified with the LPS-induced acute septic shock.3.The binding dissociation constant of SN10 is determined by BIAcore.The capacity of peptide inhibit TNF-?binding to TNFR1 competively is analyzed by ELISA and BIAcore.4.We use TNF-?stimulate HEK-293 A cells as cell model in vitro.The expression of pro-inflammatory cytokine were determined by RT-PCR.The effects on NF?B and MAPK pathway were detected by Western Blotting.We also evaluate its anti-TNF effects in DSS-induced acute colitis model in mice.5.Construction of a pet28a-SN10-Fc fusion expression vector and pc DNA3.1-SN10-Fc fusion expression vector.The protein expressed in E.coli was subjected to ion exchange chromatography for purification.The protein expressed in HEK-293 T cells was analyzed by ELISA after collection.The anti-cytotoxicity effect of the recombinant protein was evaluated by MTT assay.6.We connect the SN10 with the sequence ofamino acidsfrom known anti-TNF peptide(WSQYL).The anti-TNF effect of connective peptide is analyzed by some experiments recommended above.Results 1.The peptide contain the sequence DEQHLETELH(named as SN10)had more stronger anti-TNF effect than SN1.The peptide SN10 can also improve the survival rate of acute LPS-induced shock model in mice.2.The binding dissociation constant of SN10 is 2.775?M.SN10 cann't inhibit TNF-? binding to TNFR1 with ELISA.3.Western Blot assays revealed that SN10 inhibited the IKB,ERK,P38 phosphorylation in a dose-dependent manner in the range of 5-20?M.But the phosphorylation of JNK show no significant change.4.The treatment of SN10 ameliorates DSS-induced colitis in mice.The difference of mean colon lengths and the spleen weight/body weight index between model group and SN10-treated group is remarkable.The m RNA expression of inflammatory mediators in colon tissue is inhibited by SN10.5.We construct the pet28a-SN10-Fc fusion expression vector and pc DNA3.1-SN10-Fc fusion expression vector.The protein expressed in 293 T cells can inhibit cytotoxicity in the range of 1-5?g/ml.The protein expressed in E.coli show no remarkable effect below the concentration of 100?g/ml.6.We connect SN10 with the sequence WSQYL.We named the new peptide as SN12.It had stronger anti-TNF effect than SN10 and WSQYL.It inhibit the interaction between TNF-? and TNFR.SN12 can significantly reduced TNF-?-induced IL-8 and ICAM expression in endothelial cell.It also inhibited TNF-?-mediated activation of NF-?B and MAPKs signaling in HEK-293 A cells in a dose-dependent manner in the range of 30-120?M.Conclution The peptide isolated from T7 phage display library of Hydrophis cyanocinctus had the core sequence binding to TNFR1.SN10 revealed stronger binding capacity than SN1 in molecule level.It inhibit the TNF-?-induced IKB,ERK,P38 phosphorylation in 293 A cells and attentued the DSS-induced colitis in mice.Then we connect SN10 with the sequence WSQYL or Fc region from human immunoglobulin.The new peptide show more anti-TNF effect.The study expands our understanding of the anti-inflammatory mechanism of the venom of sea snake.It is also an attempt to find and optimize TNFR1 inhibitors.